Apoptosis or programmed cell death is the innate mechanism by which the organism eliminates unwanted cells. In contrast to necrosis, apoptosis is the most common physiological form of cell death and occurs during embryonic development, tissue remodeling, immune regulation and tumor regression. Cells undergoing apoptosis show a sequence of cardinal morphological features including membrane blebbing, cellular shrinkage and condensation of chromatin. Biochemically, these alterations are associated with the translocation of phosphatidylserine to the outer leaflet of the plasma membrane and the activation of an endonuclease which cleaves genomic DNA into multiples of internucleosomal fragments. In contrast, necrosis is classically induced following traumatic injury or exposure to high concentrations of noxious agents. Irreversible damage of the plasma membrane, mitochondrial dysfunction and cell lysis are characteristic for necrotic cell death.
Higher organisms have developed several mechanisms to rapidly and selectively eliminate cells by apoptosis. A fine-tuned mechanism to regulate life and death of a cell is the interaction of surface receptors with their cognate ligands. Several receptors are able to transmit cytotoxic signals into the cytoplasm, but in most cases they have a wide range of other functions unrelated to cell death, such as induction of cell activation, differentiation and proliferation. Whether the signals induced by a given receptor lead to cell activation or death is highly cell-type specific and tightly regulated during differentiation. For example, TNF receptors can exert co-stimulatory signals for proliferation of naive lymphocytes as well as inducing death signals required for deletion of activated lymphocytes.
Many receptors with important functions in differentiation, survival and cell death belong to an emerging family of structurally related molecules, called the TNF receptor superfamily. For some members of the family an apoptosis-inducing activity has been reported. However, most of them also have other functions such as induction of proliferation, differentiation, immune regulation and gene expression. Receptors with pleiotropic functions include TNF-R1, TNF-R2, NGF-R, CD27, CD30, CD40, OX-40, NGF-R, TRAMP (DR3/ws1-1/APO-3/LARD), HVEM (ATAR/TR2), GITR and RANK Anderson, D. M., et al., Nature 390, 175–179 (1997); Bodmer, J. L. et al., Immunity 6, 79–88 (1997); Nocentini, G. et al., Proc. Natl. Acad. Sci. 94, 6216–6221 (1997)). These receptors are type I membrane proteins which are structurally similar. Each possesses in its extracellular domain two-six imperfect repeats of about 40 amino acids, with each of approximately six Cys residues. Their cytoplasmic domains generally lack considerable sequence similarity.
APO-1/Fas, now called CD95, was the first member of the TNF receptor superfamily described in terms of its function in apoptosis (Itoh, N. et al., Cell 66, 233–243 (1991); Oehm, A. et al., J. Biol. Chem. 267, 10709–10715 (1992)). Sequence comparison of the intracellular domain of CD95 with TNF-R1 revealed that both receptors contained a similar stretch of about 80 amino acids. This region has been designated the death domain (DD) since it enables transmission of a cytotoxic signal by both molecules (Tartaglia, L. A. et al., Cell 74, 845–853 (1993); Itoh, N. et al., J. Biol. Chem. 268, 10932–10937 (1993)). Recent similarity searches in EST databases led to the cloning of a number of novel membrane receptors that contain such a death domain and are therefore referred to as the death receptors (DRs). TRAMP (DR3/ws1-1/APO-3/LARD), is both structurally and functionally similar to TNF-R1 and is abundantly expressed in T-lymphocytes (Bodmer, J. L. et al., Immunity 6, 79–88 (1997)). TRAIL-R1 (DR4, APO-2) and TRAIL-R2(DR5) have been found as receptors binding to a novel cytokine, called TRAIL for TNF-related apoptosis-inducing ligand. The two TRAIL receptors are functionally similar to CD95 as their main function seems to be to induce apoptosis (Pan, G. et al, Science 276, 111–113 (1997)). The TRAIL system, in addition, consists of two neutralizing decoy receptors, called DcR1 (TRAIL-R3, TRID, LIT) and DcR2 (TRAIL-R4) (Pan, G. et al., Science 277, 815–818 (1997); Degli-Esposti, M. A. et al., J. Exp. Med. 186, 1165–1170 (1997)). The sequence of DcR1 encodes a protein that contains the external TRAIL-binding region as well as a stretch of amino acids that anchors the receptor to the membrane. But, unlike the other receptors, DcR1 lacks an intracellular tail needed to spark the death pathway. DcR2 is also able to find TRAIL but contains a truncated death domain. Thus, both decoy receptors will prevent TRAIL from engaging functional TRAIL receptors and thereby render cells resistant to apoptosis. Collectively, this underlines that the death domain is required to induce apoptosis triggered by the different surface receptors.
For most members of the TNF-R superfamily their cognate ligands have been identified. Four of them, CD95L, TNFα, lymphotoxin-α (LTα, TNFβ) and TRAIL bind to death receptors. It was not surprising to find that, in addition to the receptors, also the ligands display structural similarities, which are reflected by similar mechanisms of receptor recognition and triggering. The ligands recognize their receptors through a shared structure composed of anti-parallel β-sheets, arranged in a jelly roll structure. As supported by structural and biochemical data, it is believed that all active ligands consist of three identical subunits and activate their receptors by oligomerization (Eck, M. J. et al., J. Biol.. Chem. 267, 2119–2122 (1992); Jones. E. Y., Immunol. Ser. 56, 93–127 (1992); Banner, D. W. et al., Cell 73, 431–445 (1993); Dhein, J. et al., J. Immunol. 149, 3166–3173 (1992)). Another common feature of the ligands is that almost all of them are type II transmembrane proteins. The only exception is LTα which, although formed as a soluble protein, binds to membrane-bound LTβ and thereby also acts as a cell-bound form. Lymphotoxins can be found as homotrimers (LTα3) or heterotrimers (LTα1/β2 or LTα2/β1). The LTα homotrimer binds the TNF receptors, whereas the heterotrimers bind to the LTβ receptor which does not contain a death domain. Although TNF-related ligands are synthesized as membrane-bound molecules, most of them also exist as soluble forms. The secreted forms are generated by rather specific metalloproteases. For TNF, a zinc-dependent metalloprotease, called TACE (TNFα-converting enzyme) was recently cloned and shown to specifically cleave TNF (Black, R. A. et al., Nature 385, 729–733 (1997); Moss, M. L. et al., Nature 385, 733–736 (1997)).
Death Receptor-Associating Proteins
A major progress in the understanding of death receptor signaling was the definition of the so-called death domain (DD), an intracellular region of about 80 amino acids that is essential for triggering cell death. Delineation of the DD was not only a major aid for the identification of new adaptor molecules when used as a bait in interactive cloning approaches. The DD exerts its effects via interactive properties, as it can self-associate and bind to the DD of other proteins. These associations between DDs occur as a consequence of receptor-ligand binding and seem to involve electrostatic interactions. As assessed by NMR spectroscopy, the DD of CD95 comprises a series of antiparallel amphipathic α-helices with many exposed charged residues (Huang, B. et al., Nature 384, 638–641 (1996)), although it should be noted that this structure was determined at acidic pH. The tendency of the DD to self-associate apparently strengthens the interactions of the receptors imposed by ligand binding. Following self-association, the DD of the receptors recruits other DD-containing proteins which then serve as adapters in the signaling cascades.
The first DD-containing adaptor proteins identified were FADD (MORT1) (Chinnaiyan et al., Cell, 81:505–512 (1995); Boldin et al., J. Biol. Chem., 270:7795–7798 (1995)), RIP (Stanger et al., Cell, 81:513–523 (1995)) and TRADD (Hsu et al., Cell, 81:495–504 (1995)). TRADD is most effectively bound following ligation of TNF-R1 where it then probably serves to recruit the DD proteins FADD and RIP as well as the RING domain adaptor protein TRAF2, FADD, in contrast, is preferentially recruited to CD95. Thus, the DD of FADD can bind to the DD of TRADD and the DD of RIP to the DDs of both TRADD and FADD. These mutual interactions may account for a potential crosstalk of the different receptor signaling pathways.
Overexpression of most DD proteins causes cell death, indicating that these molecules are involved in apoptosis signaling. In the case of FADD, transient expression of the N-terminal region was sufficient to cause apoptosis. (Chinnaiyan et al., Cell, 81:505–512 (1995)). This part of FADD was therefore termed the death effector domain (DED). In contrast, overexpression of the C-terminal DD-containing part, lacking the DED (FADD-DN), protected cells from CD95-mediated apoptosis and functioned as a dominant-negative mutant. This suggested that the N-terminus of FADD is coupled to the cytotoxic machinery. Both TRADD and RIP induce apoptosis but can also activate NF-κB, which is a typical feature of TNF-induced signaling. (Hsu et al., Cell, 81:495–504 (1995)). Similar to FADD, the C-terminus of TRADD contains a DD enabling self-association and association with the DD of other signaling molecules including TNF-R1 and FADD. TRADD, however, lacks the typical DED present in FADD.
While most of the information regarding death pathways has been obtained from yeast two-hybrid assays or supra-physiological overexpression in mammalian cells, for CD95 the signaling complexes have also been identified in vivo using classical biochemical methods. (Kischkel et al., EMBO J., 14:5579–5588 (1995)). Treatment of cells with agonistic anti-APO-1 antibodies and subsequent co-immunoprecipitation of CD95 resulted in the identification of four cytotoxicity-dependent APO-1-associated proteins (CAP1–4) on two-dimensional gels, within seconds after receptor triggering. Together with the receptor, these proteins formed the death-inducing signaling complex (DISC). Two spots were identified as two different serine-phosphorylated species of FADD, and it was demonstrated that FADD bound to CD95 in a stimulation-dependent fashion.
Sequencing of the other immunoprecipitated proteins resulted in the identification of a downstream molecule which contained two DEDs at its N-terminus that associate with the DED of FADD. (Muzio et al., Cell, 85:817–827 (1996)). At its C-terminus it had the typical domain structure of a protease like interleukin-1β converting enzyme (ICE) and was therefore termed FLICE (FADD-like ICE). FLICE was also cloned by two other groups and named MACH and Mch5. (Srinivasula et al., Proc. Natl. Acad. Sci. USA, 93:14486–14491 (1996); Alnemri et al., Cell, 87:171 (1996)). It belongs to cysteine proteases of the caspase family and is therefore now referred to as caspase-8 (Alnemri. et al., Cell, 87:171 (1996)).
Caspases have been found in organisms ranging from C. elegans to humans. Caspases were implicated in apoptosis with the discovery that CED-3, the product of a gene required for cell death in the nematode Caenorhabditis elegans, is related to mammalian interleukin-1[beta]-converting enzyme (ICE or caspase-1). (J. Yuan et al., Cell, 75:641 (1993); Thornberry et al., Nature, 356:768 (1992)). Although caspase-1 has no obvious role in cell death, it has become the first identified member of a large family of proteases whose members have distinct roles in inflammation and apoptosis. In apoptosis, caspases function in both cell disassembly (effectors) and in initiating this disassembly in response to proapoptotic signals (initiators).
Caspases share similarities in amino acid sequence, structure, and substrate specificity. (Nicholson et al., Trends Biochem. Sci., 22:299 (1997)). They are all expressed as proenzymes (30 to 50 kD) that contain three domains: an NH2-terminal domain, a large subunit (˜20 kD), and a small subunit (˜10 kD). Activation involves proteolytic processing between domains, followed by association of the large and small subunits to form a heterodimer. Crystal structures of two active caspases (caspase-1 and caspase-3) have been determined: in both cases, two heterodimers associate to form a tetramer, with two catalytic sites that appear to function independently. (Walker et al., Cell, 78:343 (1994); Wilson et al., Nature, 370:270 (1994); Rotonda et al., Nature Struct. Biol., 3:619 (1996)). Within each catalytic domain, the large and small subunits are intimately associated, with both contributing residues necessary for substrate binding and catalysis.
Two features of the proenzyme structure are central to the mechanism of activation of these enzymes. First, the NH2-terminal domain, which is highly variable in sequence and length, is involved in regulation of activation. Second, all domains are derived from the proenzyme by cleavage at caspase consensus sites, implying that these enzymes can be activated either autocatalytically or in a cascade by enzymes with similar specificity.
Caspases are among the most specific of proteases, with an unusual and absolute requirement for cleavage after aspartic acid (The only other eukaryotic protease known to have a similar specificity is the serine protease granzyme B, a mediator of granule-dependent cytotoxic T lymphocyte-mediated apoptosis). Recognition of at least four amino acids NH2-terminal to the cleavage site is also a necessary requirement for efficient catalysis. The preferred tetrapeptide recognition motif differs significantly among caspases and explains the diversity of their biological functions. (Thornberry et al., J. Biol. Chem., 272:17907 (1997)). Their specificity is even more stringent: not all proteins that contain the optimal tetrapeptide sequence are cleaved, implying that tertiary structural elements may influence substrate recognition. Cleavage of proteins by caspases is not only specific, but also highly efficient (kcat/Km>106 M−1 s−1). The strict specificity of caspases is consistent with the observation that apoptosis is not accompanied by indiscriminate protein digestion; rather, a select set of proteins is cleaved in a coordinated manner, usually at a single site, resulting in a loss or change in function.
As stated, apoptotic events include DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and disassembly into membrane-enclosed vesicles (apoptotic bodies). In vivo, this process culminates with the engulfment of apoptotic bodies by other cells, preventing complications that would result from a release of intracellular contents. These changes occur in a predictable, reproducible sequence and can be completed within 30 to 60 min. Current research suggests that a subset of caspases (effectors) is responsible for the cellular changes that occur during apoptosis and provide insights into the mechanisms that they employ.
One role of caspases is to inactivate proteins that protect living cells from apoptosis. A clear example is the cleavage of ICAD/DFF45 (Enari et al., Nature, 391:43 (1998); Liu et al., Cell, 89:175 (1997)), an inhibitor of the nuclease responsible for DNA fragmentation, CAD (caspase-activated deoxyribonuclease). In nonapoptotic cells, CAD is present as an inactive complex with ICAD. During apoptosis, ICAD is inactivated by caspases, leaving CAD free to function as a nuclease. This system is not as simple as it appears: CAD synthesized in the absence of ICAD is not active, implying that the CAD-ICAD complex is formed co-translationally, and that ICAD is required for both the activity and inhibition of this nuclease.
Other negative regulators of apoptosis cleaved by caspases are Bcl-2 proteins. (Xue et al., Nature, 390:305 (1997); Cheng et al., Science, 278:1966 (1997); Cory ibid. 281:1322 (1998)). It appears that cleavage not only inactivates these proteins, but also produces a fragment that promotes apoptosis. That such positive feedbacks are involved in the control of apoptosis is not surprising, given their importance in the regulation of other proteolytic systems.
Caspases contribute to apoptosis through direct disassembly of cell structures, as illustrated by the destruction of nuclear lamina (Takahashi et al., Proc. Natl. Acad. Sci. U.S.A., 93:8395 (1996); Orth et al., J. Biol. Chem., 271:16443 (1996)), a rigid structure that underlies the nuclear membrane and is involved in chromatin organization. Lamina is formed by head-to-tail polymers of intermediate filament proteins called lamins. During apoptosis, lamins are cleaved at a single site by caspases, causing lamina to collapse and contributing to chromatin condensation.
Caspases also reorganize cell structures indirectly by cleaving several proteins involved in cytoskeleton regulation, including gelsolin (S. Kothakota et al., Science, 278:294 (1997)), focal adhesion kinase (FAK) (Wen et al., J. Biol. Chem. 272:26056 (1997)), and p21-activated kinase 2 (PAK2). Cleavage of these proteins results in deregulation of their activity. For example, in the case of gelsolin (a protein that severs actin filaments in a regulated manner), caspase cleavage generates a fragment that is instead constitutively active.
Dissociation of regulatory and effector domains is a hallmark of caspase function. For example, they inactivate or deregulate proteins involved in DNA repair (such as DNA-PKcs), mRNA splicing (such as U1-70K), and DNA replication (such as replication factor C). Although the relationship of these cleavages to cell death is not clearly understood, it is likely that the disabling of critical homeostatic and repair functions facilitates cellular disassembly.
The observations that caspase precursors are constitutively expressed in living cells (even in neurons that can live for a lifetime) but that apoptosis can be induced quickly indicates that caspase regulation is sophisticated and effective. Complex proteolytic systems often involve a combination of regulatory proteases, cofactors, feedbacks, and thresholds that converge to control the activity of an effector protease, that in turn carries out the function of the whole process. (Beltrami et al., Proc. Natl. Acad. Sci. U.S.A., 92:8744 (1995)). This intricate regulation accounts for a spectacular feature of these systems: they keep the effector protease inactive but are able to rapidly activate large amounts of it in response to minute quantities of an appropriate inducer. Given the function of caspases as mediators of cell death, the complexity of their regulation is likely to rival that of the coagulation and complement systems.
Activation of effector caspases. A large body of genetic and biochemical evidence supports a cascade model for effector caspase activation: a proapoptotic signal culminates in activation of an initiator caspase which, in turn, activates effector caspases, resulting in cellular disassembly. Different initiator caspases mediate distinct sets of signals. For example, caspase-8 is associated with apoptosis involving death receptors. (Ashkenazi et al., Science, 281:1305 (1998)). In contrast, caspase-9 is involved in death induced by cytotoxic agents. (Hakem et al., Cell, in press; Kuida et al., ibid., in press). This model explains how distinct apoptotic signals induce the same biochemical and morphological changes.
Activation of initiator caspases. The available evidence indicates that activation of initiator caspases requires binding to specific cofactors, a mechanism commonly observed with proteases. This binding is triggered by a proapoptotic signal and mediated through one of at least two distinct structural motifs that reside in both the caspase prodomain and its corresponding cofactor. Activation of procaspase-8 requires association with its cofactor FADD (Fas-associated protein with death domain) through the DED (death effector domain) Boldin et al. ibid., 85:803 (1996); Muzio et al., ibid., p. 817), while procaspase-9 activation involves a complex with the cofactor APAF-1 through the CARD (caspase recruitment domain) (Li et al., ibid., 91:479 (1997)). Activation of caspase-9 also requires cytochrome c and deoxyadenosine triphosphate, indicating that caspase activation may require multiple cofactors.
Thus there exists a clear need for identifying and exploiting novel apoptosis related proteins. Although structurally related, such proteins may possess diverse and multifaceted functions in a variety of cell and tissue types. The inventive purified apoptosis related polypeptides are research tools useful for the identification, characterization and purification of additional proteins involved in apoptosis. Furthermore, the identification of new apoptosis related polypeptides permits the development of a range of derivatives, agonists and antagonists at the nucleic acid and protein levels which in turn have applications in the treatment and diagnosis of a range of conditions such as cancer, inflammation, neurological disorders and aberrant cell growth, amongst many other conditions.